Neural circuit-wide analysis of changes to gene expression during deafening-induced birdsong destabilization
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**eLife assessment
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This is an important study that uses the song system in a bird model to understand the transcriptional mechanisms underlying neuronal adaptations to sensory deprivation. The manuscript offers compelling data in support of their hypothesis that these transcriptional changes are related to song plasticity. The work will be of interest to biologists who study neuronal plasticity mechanisms.
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Abstract
Sensory feedback is required for the stable execution of learned motor skills, and its loss can severely disrupt motor performance. The neural mechanisms that mediate sensorimotor stability have been extensively studied at systems and physiological levels, yet relatively little is known about how disruptions to sensory input alter the molecular properties of associated motor systems. Songbird courtship song, a model for skilled behavior, is a learned and highly structured vocalization that is destabilized following deafening. Here, we sought to determine how the loss of auditory feedback modifies gene expression and its coordination across the birdsong sensorimotor circuit. To facilitate this system-wide analysis of transcriptional responses, we developed a gene expression profiling approach that enables the construction of hundreds of spatially-defined RNA-sequencing libraries. Using this method, we found that deafening preferentially alters gene expression across birdsong neural circuitry relative to surrounding areas, particularly in premotor and striatal regions. Genes with altered expression are associated with synaptic transmission, neuronal spines, and neuromodulation and show a bias toward expression in glutamatergic neurons and Pvalb/Sst- class GABAergic interneurons. We also found that connected song regions exhibit correlations in gene expression that were reduced in deafened birds relative to hearing birds, suggesting that song destabilization alters the inter-region coordination of transcriptional states. Finally, lesioning LMAN, a forebrain afferent of RA required for deafening-induced song plasticity, had the largest effect on groups of genes that were also most affected by deafening. Combined, this integrated transcriptomics analysis demonstrates that the loss of peripheral sensory input drives a distributed gene expression response throughout associated sensorimotor neural circuitry and identifies specific candidate molecular and cellular mechanisms that support the stability and plasticity of learned motor skills.
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Author Response
Reviewer #3 (Public Review):
In this study, the authors probe the molecular changes that occur in a neural circuit for learned behavior that depends on sensory input to maintain stereotypy. Songbirds, as the Bengalese finches used here are, are premier systems in which to ask these questions because they produce a highly stereotyped song that emerges after sensory learning becomes integrated into the function of a sensorimotor neural circuit responsible for singing. By deafening a group of birds (who show a shift in their song structure) and comparing them to hearing birds, clues as to how plasticity in motor output may emerge from genomic changes that alter the function of cells within the various components of the neural circuit.
There are multiple strengths of the paper:
- The results may have broad implications …
Author Response
Reviewer #3 (Public Review):
In this study, the authors probe the molecular changes that occur in a neural circuit for learned behavior that depends on sensory input to maintain stereotypy. Songbirds, as the Bengalese finches used here are, are premier systems in which to ask these questions because they produce a highly stereotyped song that emerges after sensory learning becomes integrated into the function of a sensorimotor neural circuit responsible for singing. By deafening a group of birds (who show a shift in their song structure) and comparing them to hearing birds, clues as to how plasticity in motor output may emerge from genomic changes that alter the function of cells within the various components of the neural circuit.
There are multiple strengths of the paper:
- The results may have broad implications because the type of sensorimotor neural circuit (cortico-basal ganglia-thalamic-cortical loop) used for singing is generally necessary for learned behaviors.
- The methods and analyses are generally rigorous, including the parsing of song elements, and the type of detailed RNA sequencing and analysis that demonstrates the power of a genomic view of neural plasticity as it relates to behavioral plasticity.
- Because the authors assayed the pallial (cortical) areas, as well as the basal ganglia component, of the sensorimotor circuit they were able to creatively compare how different facets of the network contributed to a) unmodified brain properties, b) properties perturbed after the loss of the auditory input that is required to stabilize song structure. As a result, they have added to the known molecular profiles for each of these brain areas, the accounting of how they may be specialized in comparison to the surrounding non-song brain, and what changes occur after deafening. Utilizing some existing single-cell sequencing data, the authors present for the first time some insight into what cell types may be showing the most robust changes, and therefore which may be driving the shift in song structure. The analysis further pushes in new ways to suggest how the molecular properties of a given brain area may relate to those of directly-connected areas. Together, these findings provide valuable clues as to the specific cell types and signaling properties that may be central to the production of stabilized, learned behavior.
- One of the cortical brain areas, LMAN, was lesioned in a subset of the hearing subjects because it projects to the area that showed the greatest molecular difference between deafened and hearing birds (RA). The idea was to compare how this affected molecular properties with properties after the loss of auditory input; because RA is the output motor area for the song, its properties may be most directly tied to song structure. Using unilateral lesions was a strong choice of experimental design that allowed for rigorous analysis of this idea, and was interpretable because birds do not have a direct inter-hemispheric callosum.
The foundation of the paper is solid, though the results shown raise several questions that are not fully addressed, and limit some of the power of the implications.
The biggest questions arise from the finding that RA shows the largest number of molecular changes after deafening. The analysis and interpretations do not fully incorporate what we know of this circuit, at least from another well-studied songbird, the zebra finch, from which the authors derive other types of information. For example, it is not yet clear if RA is most changed because it is most directly involved in song output or because it receives projections from two areas within the sensorimotor circuit (LMAN and HVC). How do we consider the fact that by adulthood, LMAN and HVC cells project onto the same RA neurons? Are those the cell types being identified here? Would HVC lesions be expected to have the same effect as LMAN lesions? Are the cell types showing the greatest change those that are most involved in song output (e.g. are they projecting to nXIIts)? How do these results relate to the findings of changes in RA after HVC and LMAN lesions reported decades ago? How do these findings compare to an earlier study that also performed sequencing on areas from the sensorimotor circuit in deafened juveniles? Further, RA also receives information from the auditory processing regions of the brain, via the immediate structure RA-cup. It is not yet explicitly addressed how some effects may be from the loss of this more direct access to auditory information, rather than from information and projections originating within the sensorimotor circuit, and reinforces the question of whether or not the number of inputs to a particular brain area is a driving factor in the general pattern of changed RNAs after perturbation.
We thank the reviewer for their review and for their excellent suggestions on how to improve its impact. The reviewer raises several important points, which we have expanded on in the Discussion of the revised manuscript, and will address here:
First, there is the general consideration of how the structure of inputs to RA influences the interpretation of our results. There is the question about whether we can consider RA expression alterations as due to its direct projections to song motoneurons (‘output’) or the convergence of two important song nuclei, HVC and LMAN, onto RA (‘input’). This is a difficult question to untangle. We could interpret ‘output’ only effects as local perturbations that do not depend on song circuit afferent activity, such as hormonal fluctuations associated with the loss of hearing. ‘Input’ effects would occur through changes in afferent activity, such as those that elicit plasticity associated with song destabilization or more general alterations to the amount of afferent neural activity (a point addressed in the revised manuscript, lines 842-848). By focusing on a measure of song destabilization in our differential expression analysis, we are specifically seeking to identify gene expression responses that are associated with changes to behavioral output. Yet these behavioral changes are certainly driven by alterations in upstream regions or the manner in which they converge onto RA. The reviewer also notes inputs from RA-cup as a potential avenue through which the loss of auditory information could more directly influence expression in RA. It is certainly possible that the loss of auditory information itself could influence gene expression in different components of the song system, a point we note in the revised Discussion (lines 848-853). We also note there that future experiments leveraging different plasticity induction techniques (TS cut, delayed auditory feedback) will be important to resolve the influence of this input.
Our lesion experiments aimed to characterize how input from LMAN influences expression in RA, due to LMAN’s important role in mediating song plasticity. We would expect HVC lesions to elicit different expression responses because of its distinct mode of transmission onto RA projection neurons (primarily AMPAR in contrast to primarily NMDAR for LMAN), the distinct activity patterns of HVC and LMAN, and likely distinct neuromodulatory signaling from the two afferents (e.g. LMAN acts as source of BDNF). We discuss how HVC lesions would be useful to further disentangle the influence of afferent input on RA gene expression in the Discussion of the revised manuscript (lines 926-946). In the revised manuscript, we also cite previous work that examined the influence of HVC and LMAN on RA neural activity, morphology, and cell survival (lines 928-932).
As to the cell types in RA that show expression changes following deafening, we show in Figure 5 that both glutamatergic projection neurons (‘RA_Glut’), i.e. the neurons that project to subcerebral structures such as nXIIts, as well as GABAergic interneurons (‘GABA’) show substantial expression alterations. In the Discussion, we highlight the functional roles of several genes that have enriched expression in each class (lines 864-873 and 887-893).
In the revised manuscript, we have added a paragraph in the Discussion (lines 854-862) that references results from Mori, C. & Wada, K. Audition-independent vocal crystallization associated with intrinsic developmental gene expression dynamics. J. Neurosci. 35, 878–889 (2015). This work examined the influence of early deafening on gene expression in the song motor pathway and identified a strong developmental and audition-independent expression response. It identified an important separation between developmentally-driven and experience-dependent molecular responses in the song system. We note that the aims were distinct from the present study, which sought to identify gene expression responses to deafening-induced song plasticity.
Importantly, since the LMAN lesions did not create significant changes in the song structure, it is difficult to know how to interpret the meaning of these molecular changes in RA, alone and in combination with the comparison to the RA profiles from deafened birds. Of importance is the question of whether or which molecular profiles are thus signatures of behavioral plasticity or not.
The reviewer raises an important set of followup experiments that assess the extent to which the transcriptional state of the song system tracks with song plasticity state. Coupling LMAN lesions with deafening, a manipulation that prevents song degradation, would be a strong approach to identify genes whose expression is closely tied to song destabilization, a possibility that we now discuss (lines 936-946).
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**eLife assessment
**
This is an important study that uses the song system in a bird model to understand the transcriptional mechanisms underlying neuronal adaptations to sensory deprivation. The manuscript offers compelling data in support of their hypothesis that these transcriptional changes are related to song plasticity. The work will be of interest to biologists who study neuronal plasticity mechanisms. -
Reviewer #1 (Public Review):
In this manuscript, the authors seek to define the transcriptional response to deafening in the songbird brain. They compare transcriptional changes in the song regions with changes in the non-singing-associated surrounds, compute a song degradation score against which they can compare gene expression, and they use single-cell sequencing data from these brain regions to map genes to cells. The study is impressively comprehensive for time points, replicates, brain regions, comparisons, and alternative strategies (e.g. the LMAN lesions). This dataset builds nicely upon studies that assessed gene expression changes upon singing and applies a broad and useful series of bioinformatics analyses to get the strongest evidence for function from the data.
I think this dataset will be of great interest to a broad range …
Reviewer #1 (Public Review):
In this manuscript, the authors seek to define the transcriptional response to deafening in the songbird brain. They compare transcriptional changes in the song regions with changes in the non-singing-associated surrounds, compute a song degradation score against which they can compare gene expression, and they use single-cell sequencing data from these brain regions to map genes to cells. The study is impressively comprehensive for time points, replicates, brain regions, comparisons, and alternative strategies (e.g. the LMAN lesions). This dataset builds nicely upon studies that assessed gene expression changes upon singing and applies a broad and useful series of bioinformatics analyses to get the strongest evidence for function from the data.
I think this dataset will be of great interest to a broad range of researchers who study neuronal plasticity mechanisms.
-
Reviewer #2 (Public Review):
Like humans, Bengalese finches rely on auditory feedback to maintain the acoustic stability of their learned vocalizations, and deafening causes acoustic degradation of their songs. How disruptions to sensory input alter gene expression in brain regions important for singing and song learning remains relatively unexplored. The authors develop an innovative serial laser capture RNA-sequencing method, which allows them to conduct large-scale analyses of gene expression in spatially defined singing-related regions, as well as in surrounding non-singing-related regions. These methods are used to demonstrate that deafening preferentially alters gene expression in song-related regions relative to surrounding song-related areas, and that deafening reduces correlations in gene expression between connected …
Reviewer #2 (Public Review):
Like humans, Bengalese finches rely on auditory feedback to maintain the acoustic stability of their learned vocalizations, and deafening causes acoustic degradation of their songs. How disruptions to sensory input alter gene expression in brain regions important for singing and song learning remains relatively unexplored. The authors develop an innovative serial laser capture RNA-sequencing method, which allows them to conduct large-scale analyses of gene expression in spatially defined singing-related regions, as well as in surrounding non-singing-related regions. These methods are used to demonstrate that deafening preferentially alters gene expression in song-related regions relative to surrounding song-related areas, and that deafening reduces correlations in gene expression between connected song-related regions. The authors then compare their findings to a previous single-cell RNA sequencing dataset to determine the cell types whose gene expression is likely to be most strongly affected by deafening and song degradation. Finally, the authors repeat their measurements of gene expression changes in RA following unilateral lesion of LMAN and find that LMAN lesions have the largest effect on groups of genes whose expression was also strongly affected by deafening. The study is elegant and rigorous, and its conclusions are well-supported. This work reveals candidate genes that may play a role in stable vocal performance and whose changes in expression may contribute to the acoustic degradation of vocal performance following deafening.
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Reviewer #3 (Public Review):
In this study, the authors probe the molecular changes that occur in a neural circuit for learned behavior that depends on sensory input to maintain stereotypy. Songbirds, as the Bengalese finches used here are, are premier systems in which to ask these questions because they produce a highly stereotyped song that emerges after sensory learning becomes integrated into the function of a sensorimotor neural circuit responsible for singing. By deafening a group of birds (who show a shift in their song structure) and comparing them to hearing birds, clues as to how plasticity in motor output may emerge from genomic changes that alter the function of cells within the various components of the neural circuit.
There are multiple strengths of the paper:
1. The results may have broad implications because the type of …Reviewer #3 (Public Review):
In this study, the authors probe the molecular changes that occur in a neural circuit for learned behavior that depends on sensory input to maintain stereotypy. Songbirds, as the Bengalese finches used here are, are premier systems in which to ask these questions because they produce a highly stereotyped song that emerges after sensory learning becomes integrated into the function of a sensorimotor neural circuit responsible for singing. By deafening a group of birds (who show a shift in their song structure) and comparing them to hearing birds, clues as to how plasticity in motor output may emerge from genomic changes that alter the function of cells within the various components of the neural circuit.
There are multiple strengths of the paper:
1. The results may have broad implications because the type of sensorimotor neural circuit (cortico-basal ganglia-thalamic-cortical loop) used for singing is generally necessary for learned behaviors.2. The methods and analyses are generally rigorous, including the parsing of song elements, and the type of detailed RNA sequencing and analysis that demonstrates the power of a genomic view of neural plasticity as it relates to behavioral plasticity.
3. Because the authors assayed the pallial (cortical) areas, as well as the basal ganglia component, of the sensorimotor circuit they were able to creatively compare how different facets of the network contributed to a) unmodified brain properties, b) properties perturbed after the loss of the auditory input that is required to stabilize song structure. As a result, they have added to the known molecular profiles for each of these brain areas, the accounting of how they may be specialized in comparison to the surrounding non-song brain, and what changes occur after deafening. Utilizing some existing single-cell sequencing data, the authors present for the first time some insight into what cell types may be showing the most robust changes, and therefore which may be driving the shift in song structure. The analysis further pushes in new ways to suggest how the molecular properties of a given brain area may relate to those of directly-connected areas. Together, these findings provide valuable clues as to the specific cell types and signaling properties that may be central to the production of stabilized, learned behavior.
4. One of the cortical brain areas, LMAN, was lesioned in a subset of the hearing subjects because it projects to the area that showed the greatest molecular difference between deafened and hearing birds (RA). The idea was to compare how this affected molecular properties with properties after the loss of auditory input; because RA is the output motor area for the song, its properties may be most directly tied to song structure. Using unilateral lesions was a strong choice of experimental design that allowed for rigorous analysis of this idea, and was interpretable because birds do not have a direct inter-hemispheric callosum.
The foundation of the paper is solid, though the results shown raise several questions that are not fully addressed, and limit some of the power of the implications.
The biggest questions arise from the finding that RA shows the largest number of molecular changes after deafening. The analysis and interpretations do not fully incorporate what we know of this circuit, at least from another well-studied songbird, the zebra finch, from which the authors derive other types of information. For example, it is not yet clear if RA is most changed because it is most directly involved in song output or because it receives projections from two areas within the sensorimotor circuit (LMAN and HVC). How do we consider the fact that by adulthood, LMAN and HVC cells project onto the same RA neurons? Are those the cell types being identified here? Would HVC lesions be expected to have the same effect as LMAN lesions? Are the cell types showing the greatest change those that are most involved in song output (e.g. are they projecting to nXIIts)? How do these results relate to the findings of changes in RA after HVC and LMAN lesions reported decades ago? How do these findings compare to an earlier study that also performed sequencing on areas from the sensorimotor circuit in deafened juveniles? Further, RA also receives information from the auditory processing regions of the brain, via the immediate structure RA-cup. It is not yet explicitly addressed how some effects may be from the loss of this more direct access to auditory information, rather than from information and projections originating within the sensorimotor circuit, and reinforces the question of whether or not the number of inputs to a particular brain area is a driving factor in the general pattern of changed RNAs after perturbation.
Importantly, since the LMAN lesions did not create significant changes in the song structure, it is difficult to know how to interpret the meaning of these molecular changes in RA, alone and in combination with the comparison to the RA profiles from deafened birds. Of importance is the question of whether or which molecular profiles are thus signatures of behavioral plasticity or not.
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